PROJECT SUMMARY: The hematopoietic stem cell (HSC) niche is a complex structure thought to play an important role in regulating HSC function and numbers. Among the niche constituents, MSCs express the highest levels of the major niche factors. Yet, their ability to maintain HSCs ex vivo has been limited thus far. MSCs indeed rapidly loose expression of major niche factors in culture. We have tested the hypothesis that MSCs? transcriptional machinery was altered in culture conditions and that reinstating the expression of key transcription regulators would revitalize niche activity. We have carried out a genetic screen using 28 transcription regulators that were expressed at high levels in Nestin-GFP+ niche cells in vivo but downregulated in culture, and identified a combination of 5 genes (Ostf1, Xbp1, Irf3, Irf7 and Klf7) that can revitalize MSCs. Revitalized MSCs (rMSCs) exhibit higher niche factor expression and capacity to expand either murine or human HSCs compared to control MSCs. Using RNA-seq and ATAC-seq analyses, we have identified Mef2c as downstream effector of the revitalization process. This continuation proposal will evaluate the hypothesis that HSCs are finely regulated by a niche programmed to maintain their numbers. In Specific Aim 1, we will assess the function of human orthologues of the 5 factors in revitalization of human MSCs. We will use stable isotope labeling with amino acids in cell culture (SILAC) and labeling by azidohomoalanine (AHA) to identify by mass spectrometry novel soluble factors derived from rMSCs. We will test the function of rMSCs in the maturation of embryonic stem (ES) cell-derived HSCs to promote their engraftment in immunodeficient mice. In Specific Aim 2, we will evaluate the role of key transcription regulators in HSC niche activity, focusing our studies on Snai2, Ostf1 and Mef2c using conditional deletions using floxed mouse lines. In Specific Aim 3, we will assess whether HSC numbers are regulated by local niche availability or systemic sensing using transplantation, local irradiation, and mathematical modeling. The proposed studies will shed new light on mechanisms by which HSC numbers are regulated and provide new methods toward their ex vivo expansion.